An immunohistochemical atlas of necroptotic pathway expression.

Necroptosis is a lytic form of regulated cell death reported to contribute to inflammatory diseases of the gut, skin and lung, as well as ischemic-reperfusion injuries of the kidney, heart and brain. However, precise identification of the cells and tissues that undergo necroptotic cell death in vivo has proven challenging in the absence of robust protocols for immunohistochemical detection. Here, we provide automated immunohistochemistry protocols to detect core necroptosis regulators - Caspase-8, RIPK1, RIPK3 and MLKL - in formalin-fixed mouse and human tissues. We observed surprising heterogeneity in protein expression within tissues, whereby short-lived immune barrier cells were replete with necroptotic effectors, whereas long-lived cells lacked RIPK3 or MLKL expression. Local changes in the expression of necroptotic effectors occurred in response to insults such as inflammation, dysbiosis or immune challenge, consistent with necroptosis being dysregulated in disease contexts. These methods will facilitate the precise localisation and evaluation of necroptotic signaling in vivo.

A common human MLKL polymorphism confers resistance to negative regulation by phosphorylation.

Across the globe, 2-3% of humans carry the p.Ser132Pro single nucleotide polymorphism in MLKL, the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. Here we show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKLS132P in biological membranes and MLKLS132P overriding pharmacological and endogenous inhibition of MLKL. In mouse cells, the equivalent Mlkl S131P mutation confers a gene dosage dependent reduction in sensitivity to TNF-induced necroptosis in both hematopoietic and non-hematopoietic cells, but enhanced sensitivity to IFN-ß induced death in non-hematopoietic cells. In vivo, MlklS131P homozygosity reduces the capacity to clear Salmonella from major organs and retards recovery of hematopoietic stem cells. Thus, by dysregulating necroptosis, the S131P substitution impairs the return to homeostasis after systemic challenge. Present day carriers of the MLKL S132P polymorphism may be the key to understanding how MLKL and necroptosis modulate the progression of complex polygenic human disease.

MLKL deficiency protects against low-grade, sterile inflammation in aged mice.

MLKL and RIPK3 are the core signaling proteins of the inflammatory cell death pathway, necroptosis, which is a known mediator and modifier of human disease. Necroptosis has been implicated in the progression of disease in almost every physiological system and recent reports suggest a role for necroptosis in aging. Here, we present the first comprehensive analysis of age-related histopathological and immunological phenotypes in a cohort of Mlkl-/- and Ripk3-/- mice on a congenic C57BL/6 J genetic background. We show that genetic deletion of Mlkl in female mice interrupts immune system aging, specifically delaying the age-related reduction of circulating lymphocytes. -Seventeen-month-old Mlkl-/- female mice were also protected against age-related chronic sterile inflammation in connective tissue and skeletal muscle relative to wild-type littermate controls, exhibiting a reduced number of immune cell infiltrates in these sites and fewer regenerating myocytes. These observations implicate MLKL in age-related sterile inflammation, suggesting a possible application for long-term anti-necroptotic therapy in humans.

Molecular Diagnosis of Muscular Dystrophy Patients in Western Indian Population: A Comprehensive Mutation Analysis Using Amplicon Sequencing.

Muscular Dystrophies (MDs) are a group of inherited diseases and heterogeneous in nature. To date, 40 different genes have been reported for the occurrence and/or progression of MDs. This study was conducted to demonstrate the application of next-generation sequencing (NGS) in developing a time-saving and cost-effective diagnostic method to detect single nucleotide variants (SNVs) and copy number variants (CNVs) in a single test. A total of 123 cases clinically suspected of MD were enrolled in this study. Amplicon panel-based diagnosis was carried out for 102 (DMD/BMD) cases and the results were further screened using multiplex ligation-dependent probe amplification (MLPA). Whilst in the case of LGMD (N = 19) and UMD (N = 2), only NGS panel-based analysis was carried out. We identified the large deletions in 74.50% (76/102) of the cases screened with query DMD or BMD. Further, the large deletion in CAPN3 gene (N = 3) and known SNV mutations (N = 4) were identified in LGMD patients. Together, the total diagnosis rate for this amplicon panel was 70.73% (87/123) which demonstrated the utility of panel-based diagnosis for high throughput, affordable, and time-saving diagnostic strategy. Collectively, present study demonstrates that the panel based NGS sequencing could be superior over to MLPA.

Hyperuricemia and Cardiovascular Risk.

As the prevalence of hyperuricemia (elevated uric acid levels in the blood) increases, the relationship between serum uric acid levels and cardiovascular risk has garnered increased interest. Several studies have highlighted that elevated uric acid levels are likely tied to increased cardiovascular disease risk. Specifically, the presence of hyperuricemia is well-established to contribute to the onset of gout (an inflammatory condition characterized by painful/swollen joints). Several studies have shown that the risk of developing gout is strongly associated with the degree of hyperuricemia. In this review, we will provide insight into the association between gout and cardiovascular disease risk. It is also important to gain insight into the pathophysiology of gout to understand the contributions to cardiovascular disease risk as well as improve diagnosis and target treatment more effectively. An interdisciplinary approach for gout management and areas for further investigation will be discussed in this review.

Tabes Dorsalis in a Patient Presenting With Right Lower Extremity Paresthesia and Cervical Spine Pain.

Syphilitic myelitis, also known as tabes dorsalis, is a disease affecting the posterior columns of the spinal cord and dorsal roots and presents as sensory ataxia and neuropathic pain and less commonly as paresthesia and gastrointestinal disturbance. Tabes dorsalis is the clinical manifestation of a previous infection with syphilis, and the average latency period from initial infection to presentation of symptoms is approximately 25 years. This is a rarely encountered manifestation of syphilis since the widespread usage of antibiotics. Penicillin G is the mainstay therapy of neurosyphilis and has been shown to improve and resolve spinal cord lesions associated with tertiary syphilis. We present a case of tabes dorsalis in a 56-year-old female with a history of extensive autoimmune disease who initially presented with neck pain and numbness of the right lower extremity. The unique nature of this case lies in the patient's clinical course, as her symptoms were initially attributed to her history of autoimmune disease. A reactive CSF-VDRL (cerebrospinal fluid Venereal Disease Research Laboratory) test and MRI findings led clinicians to suspect neurosyphilis and begin penicillin G. The patient began to show significant clinical improvement after penicillin G therapy was begun and was discharged to a rehabilitation facility to continue antibiotics and begin aggressive physical therapy.

A toolbox for imaging RIPK1, RIPK3, and MLKL in mouse and human cells.

Necroptosis is a lytic, inflammatory cell death pathway that is dysregulated in many human pathologies. The pathway is executed by a core machinery comprising the RIPK1 and RIPK3 kinases, which assemble into necrosomes in the cytoplasm, and the terminal effector pseudokinase, MLKL. RIPK3-mediated phosphorylation of MLKL induces oligomerization and translocation to the plasma membrane where MLKL accumulates as hotspots and perturbs the lipid bilayer to cause death. The precise choreography of events in the pathway, where they occur within cells, and pathway differences between species, are of immense interest. However, they have been poorly characterized due to a dearth of validated antibodies for microscopy studies. Here, we describe a toolbox of antibodies for immunofluorescent detection of the core necroptosis effectors, RIPK1, RIPK3, and MLKL, and their phosphorylated forms, in human and mouse cells. By comparing reactivity with endogenous proteins in wild-type cells and knockout controls in basal and necroptosis-inducing conditions, we characterise the specificity of frequently-used commercial and recently-developed antibodies for detection of necroptosis signaling events. Importantly, our findings demonstrate that not all frequently-used antibodies are suitable for monitoring necroptosis by immunofluorescence microscopy, and methanol- is preferable to paraformaldehyde-fixation for robust detection of specific RIPK1, RIPK3, and MLKL signals.

Mutational Landscape for Indian Hereditary Breast and Ovarian Cancer Cohort Suggests Need for Identifying Population Specific Genes and Biomarkers for Screening.

BACKGROUND: Breast and ovarian cancers are the most prevalent cancers and one of the leading causes of death in Indian women. The healthcare burden of breast and ovarian cancers and the rise in mortality rate are worrying and stress the need for early detection and treatment. METHODS: We performed amplicon sequencing of 144 cases who had breast/ovarian cancer disease (total 137 cases are patients and seven are tested for BRCA1/2 carrier) Using our custom designed gene panel consisting of 14 genes, that are associated with high to moderate risk of breast and ovarian cancers. Variants were called using Torrent Variant Caller and were annotated using ThermoFisher's Ion Reporter software. Classification of variants and their clinical significance were identified by searching the variants against ClinVar database. RESULTS: From a total of 144 cases, we were able to detect 42 pathogenic mutations in [40/144] cases. Majority of pathogenic mutations (30/41) were detected in BRCA1 gene, while (7/41) pathogenic mutations were detected in BRCA2 gene, whereas, (2/41) pathogenic mutations were detected in TP53 gene and BRIP1, PALB2, and ATM genes respectively. So, BRCA genes contributed 88.09% of pathogenic mutations, whereas non-BRCA genes contributed 11.91% of pathogenic mutations. We were also able to detect 25 VUS which were predicted to be damaging by in silico prediction tools. CONCLUSION: Early detection of cancers in the Indian population can be done by genetic screening using customized multi-gene panels. Indications of our findings show that in the Indian population, apart from the common BRCA genes, there are other genes that are also responsible for the disease. High frequency mutations detected in the study and variants of uncertain significance predicted to be damaging by in silico pathogenicity prediction tools can be potential biomarkers of hereditary breast and ovarian cancer in Indian HBOC patients.